The production of mushroom spawn is old and well known. For example, dry grain is placed in a bottle with water and calcium carbonate and closed with a cotton plug and sterilized. After sterilization, the bottle is cooled and the grain is inoculated with mycelium, reclosed and permitted to stand for the period of mycelium incubation. U.S. Pat. No. 1,896,517. In the cultivation of mushroom spawn sterilization of the nutrients and growing containers is very necessary in order to avoid the growth of other bacteria or molds which would inhibit the mushroom spawn.
The production of tempeh also has been known, particularly in the oriental culture, for centuries. Tempeh is fermented from soybeans inoculated with several species of Rhizopus. Recent techniques permit tempeh to be rapidly fermented in plastic bags in which the mold forms a mycelial mass binding the soybeans into a cake-like product.
Improvements in growing mushroom spawn include pre-cooking and swelling the grains to prevent their caking during sterilization, U.S. Pat. No. 2,520,318 using wood charcoal granular bases having a cereal substrate coating for growing the spawn, U.S. Pat. No. 2,677,917; and using sterilizable plastic bags for incubation of mycelia and shipping thereof, U.S. Pat. No. 2,851,821.
With respect to the method described in U.S. Pat. No. 2,851,821, elongated plastic bags are filled with the desired substrate and closed using a breathable filtering material in the mouth of the bag. The bag and contents are thereafter sterilized in much the same manner as the predecessor glass bottles, or by irradiation and inoculated with the desired inoculum and incubated. The advantages of this process include the elimination of glass bottles which were costly, fragile and cumbersome and permitted all process steps to be carried out in the bag and after incubation allowed the spawn to be transported to the growing site without removing the culture or spawn to an intermediate container. Inasmuch as plants for cultivation are relatively expensive to build and operate, they are few and far between which necessitates transporting spawn considerable distances. The advent of the plastic incubation bag represented a substantial improvement in the cultivation and transportation of mushroom spawn. Further improvements to that method were also suggested for transporting spawn. For example, U.S. Pat. No. 3,335,521 taught that the champigon spawn could be shipped in an air-tight container with ice.
Even today the preferred method of inoculating and incubating mycelia takes place in flexible plastic containers. Accordingly many advancements in the design and construction of containers have been suggested including British Patent No. 1,176,188 in which the filtering element is incorporated into the wall of the container. See also U.S. Pat. No. 3,938, 658 which teaches the use of a porous filter media over slits formed in the walls of a plastic bag. And, most recently, U.S. Pat. No. 4,063,383 which teaches growing mushroom spawn in a plastic bag having a microporous breathing panel that acts as a lining for perforations in the bag.
In conventional methods of cultivating the mushroom spawn, whether using glass bottles or plastic bags, the nutrient or substrate is normally sterilized in the container to prevent the growth of unwanted bacteria or other undesired microorganisms. These methods, however, are labor intensive. The sterilization of nutrient filled bottles or bags, the inoculation of the sterilized nutrient containers and the agitation of the individual nutrient containers to mix the inoculum during incubation requires numerous manual steps. Also, it had been heard that attempts many years ago by Somycel Company in France to sterilize and inoculate the substrate in large batches prior to filling the bottles were unsuccessful and abandoned because of contamination problems.
The present invention overcomes the problems inherent in prior art methods and reduces the labor involved in the sterilization and inoculation processes as well as minimizes the possibility of contamination during inoculation and subsequent bag filling operations. It is also an object of the present invention to eliminate the mixing steps previously required after inoculation of the nurient containers. A further object of the invention is to provide a method for producing metabolites on a solid medium rather than in a liquid fermentation process. The invention is also useful in the preparation of cultures for fermentation of food products such as tempeh. In addition to the method of the present invention, it is also an object to provide apparatus for carrying out such processes. Moreover, the invention provides apparatus wherein the sterilized nutrient is bulk inoculated, blended and sterilely discharged into sterile containers for incubation. The apparatus and method of the present invention overcome many of the obstacles encountered in cultivating cell lines on solid substrate under sterile conditions.